Prenatal benzene exposure in mice alters offspring hypothalamic development predisposing to metabolic disease in later life

Abstract

Maternal exposure to environmental contaminants during pregnancy poses a significant threat to a developing fetus, as these substances can easily cross the placenta and disrupt the neurodevelopment of offspring. Specifically, the hypothalamus is essential in the regulation of metabolism, notably during critical windows of development. An abnormal hormonal and inflammatory milieu during development can trigger persistent changes in the function of hypothalamic circuits, leading to long-lasting effects on the body's energy homeostasis and metabolism. We recently demonstrated that gestational exposure to clinically relevant levels of benzene induces severe metabolic dysregulation in the offspring. Given the central role of the hypothalamus in metabolic control, we hypothesized that prenatal exposure to benzene impacts hypothalamic development, contributing to the adverse metabolic effects in the offspring. C57BL/6JB dams were exposed to benzene at 50 ppm in the inhalation chambers exclusively during pregnancy (from E0.5 to E19). Transcriptomic analysis of the exposed offspring at postnatal day 21 (P21) revealed hypothalamic changes in genes related to metabolic regulation, inflammation, and neurodevelopment exclusively in males. Moreover, the hypothalamus of prenatally benzene-exposed male offspring displayed alterations in orexigenic and anorexigenic projections, impairments in leptin signaling, and increased microgliosis. Additional exposure to benzene during lactation did not promote further microgliosis or astrogliosis in the offspring, while the high-fat diet (HFD) challenge in adulthood exacerbated glucose metabolism and hypothalamic inflammation in benzene-exposed offspring of both sexes. These findings reveal the persistent adverse effects of prenatal benzene exposure on hypothalamic circuits and neuroinflammation, predisposing the offspring to long-lasting metabolic health conditions.

Keywords: Air pollution; Hypothalamic development; Metabolic programming; Metabolic syndrome; Neuroinflammation; Prenatal exposures.

Figure 1.. Prenatal benzene exposure alters hypothalamic transcriptome in the male offspring.

(A) Experimental design of maternal benzene exposure and RNAseq of the hypothalamus of male and female offspring (n=3–5 male and female offspring per litter, from 3–4 litters per condition) (B) Average differentially expressed genes (DEGs) of both male and female offspring P21 hypothalamus. (C) Biological process and pathways of the top DEGs in male offspring (FDR< 0.01). (D) KEGG pathways enrichment of DEGs in male offspring (p<0.05) (E) Biological pathway enrichment of male offspring DEGs (p<0.05) (F) Enrichment of cellular compartments in DEGs of male offspring (p<0.05).

Figure 2.. Prenatal benzene exposure alters hypothalamic orexigenic and anorexigenic projections in male offspring.

(A) Representative images of neuronal projections in the PVH identified by immunofluorescent detection of alpha-melanocyte-stimulating hormone (α-MSH) in P21 offspring. (B) Representative images of neuronal projections in the PVH identified by immunofluorescent detection of agouti-related protein (AgRP) in P21 offspring. (C) Quantitation of α-MSH fiber density in male and female offspring at 20x magnification (n= 3–5 per group). (D) Quantitation of AgRP fiber density in male and female offspring at 20x magnification (n= 3–5 per group). Data were expressed as the mean ± SEM and analyzed by t-test (*= vs control; *p<0.05, **p<0.01).

Figure 3.. Prenatal benzene exposure alters hypothalamic leptin signaling in male offspring.

(A) Representative images of phosphorylated signal transducer and activator of transcription −3 (pSTAT3) positive cells in the arcuate nucleus (ARC) and dorsomedial hypothalamic nucleus (DMH) of P21 offspring. Quantification of pSTAT3+ cells in the ARC identified by immunofluorescence in male (B) and female (D) offspring at 20x magnification (n=3–5 per group). Quantification of pSTAT3+ cells in the DMH of male (C) and female (E) P21 offspring. Data were expressed as the mean ± SEM and analyzed by t-test if necessary to compare between only two groups of predictor variable or Two-way ANOVA followed by Newman-Keuls post hoc analysis (* = vs control; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Figure 4.. Prenatal benzene exposure induces hypothalamic microgliosis in the offspring.

(A) Representative images of microglia identified by immunofluorescent detection of ionized calcium-binding adaptor molecule 1 (Iba1) in P21 ARC at 10x magnification. (B) Representative images of astrocytes identified by immunofluorescent detection of glial fibrillary acidic protein (GFAP) in P21 ARC at 10x magnification. (C) Microglial morphology in P21 offspring of both sexes. (D) Astrocyte morphology in P21 offspring of both sexes. (E) Quantitation of Iba1 immunoreactivity (n=5 per group). (G) Quantitation of GFAP immunoreactivity (n=3–5 per group). (F; H-L) Measurement of microglial and astrocyte branch length, endpoints, and density in P21 offspring by skeleton analysis (see Methods). Data were expressed as the mean ± SEM and analyzed by t-test (*= vs control; *p<0.05).

Figure 5.. Benzene exposure during lactation does not exacerbate hypothalamic gliosis in the offspring.

(A) Experimental timeline of exposure during gestation and lactation. (B) Representative images of microglia identified by Iba1 immunofluorescence in male and female P21 offspring ARC. (C) Representative images of astrocytes identified by GFAP immunofluorescence in male and female P21 offspring ARC. Quantification of microglia (D) and astrocytes (E) in P21 offspring of both sexes at 10x magnification (n=4–5 per group). Data were expressed as the mean ± SEM and analyzed by t-test (*= vs control; **p<0.01).

Figure 6.. Prenatal benzene exposure predisposes HFD-fed offspring to susceptibility to metabolic alterations in adulthood.

(A) Timeline of experimental design. (B) Body weight (g) of adult HFD males (B) and females (C). Glucose tolerance test in 6-month-old HFD males (D) and females (E). Quantification of Iba1+ cells in the MBH of male (F) and female (H) 6-month-old offspring. Quantification of GFAP+ cells in the MBH of male (G) and female (I) 6-month-old offspring. Data were expressed as mean ± SEM (n=3–4 per group) and analyzed by t-test if necessary to compare between only two groups of predictor variable or repeated measures ANOVA followed by Newman-Keuls post hoc analysis, (*= vs control; *p<0.05).